Space flight alters bacterial gene expression and
virulence and reveals a role for global regulator Hfq
J. W. Wilsona,b, C. M. Ottc, K. Höner zu Bentrupb, R. Ramamurthyb, L. Quicka, S. Porwollikd, P. Chengd, M. McClellandd,
G. Tsaprailise, T. Radabaughe, A. Hunte, D. Fernandeza, E. Richtera, M. Shahf, M. Kilcoynef, L. Joshif,
M. Nelman-Gonzalezg, S. Hingh, M. Parrah, P. Dumarsh, K. Norwoodi, R. Boberi, J. Devichi, A. Rugglesi,
C. Goulartj, M. Rupertj, L. Stodieckj, P. Staffordk, L. Catellai, M. J. Schurrb,l, K. Buchananb,m, L. Moricib,
J. McCrackenb,n, P. Allenb,o, C. Baker-Colemanb,o, T. Hammondb,o, J. Vogelp, R. Nelsonq, D. L. Piersonc,
H. M. Stefanyshyn-Piperr, and C. A. Nickersona,b,s
aCenter for Infectious Diseases and Vaccinology, fCenter for Glycoscience Technology, kCenter for Innovations in Medicine, and qCenter for Combinatorial
Sciences, The Biodesign Institute, Arizona State University, Tempe, AZ 85287; bTulane University Health Sciences Center, New Orleans, LA 70112;
cHabitability and Environmental Factors Division and rAstronaut Office, Johnson Space Center, National Aeronautics and Space Administration,
Houston, TX 77058; dSidney Kimmel Cancer Center, San Diego, CA 92121; eCenter for Toxicology, University of Arizona, Tucson, AZ 85721; gWyle
Laboratories, Houston, TX 77058; hAmes Research Center, National Aeronautics and Space Administration, Moffett Field, CA 94035; iSpace Life
Sciences Laboratory, Kennedy Space Center, Cape Canaveral, FL 32920; jBioServe, University of Colorado, Boulder, CO 80309; lUniversity of
Colorado at Denver and Health Sciences Center, Denver, CO 80262; mOklahoma City University, Oklahoma City, OK 73106; nSection of
General Surgery, University of Chicago, Chicago, IL 60637; oSoutheast Louisiana Veterans Health Care System, New Orleans, LA 70112;
and pRNA Biology Group, Max Planck Institute for Infection Biology, 10117 Berlin, Germany
A comprehensive analysis of both the molecular genetic and
phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight
on microbial pathogenicity and associated infectious disease risks
have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and
compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73
proteins changed expression with the conserved RNA-binding
protein Hfq identified as a likely global regulator involved in the
response to this environment. Hfq involvement was confirmed
with a ground-based microgravity culture model. Space flight
samples exhibited enhanced virulence in a murine infection model
and extracellular matrix accumulation consistent with a biofilm.
Strategies to target Hfq and related regulators could potentially
decrease infectious disease risks during space flight missions and
provide novel therapeutic options on Earth.
microgravity ! Space Shuttle ! low shear modeled microgravity ! rotating
wall vessel ! Salmonella
E
nvironmental conditions and crew member immune dysfunction associated with space flight may increase the risk of
infectious disease during a long-duration mission (1–4). However, our knowledge of microbial changes in response to space
flight conditions and the corresponding changes to infectious
disease risk is limited and unclear. Elucidation of such risks and
the mechanisms behind any space flight-induced changes to
microbial pathogens holds the potential to decrease risk for
human exploration of space and provide insight into how
pathogens cause infections in Earth-based environments. Numerous logistical and technological hurdles exist when performing biological space flight experimentation, and an extremely
limited number of opportunities to perform such research are
available. Accordingly, comprehensive analysis of cells, including pathogenic microbes, at the molecular and phenotypic level
during space flight offers a rare opportunity to examine their
behavior and response in this environment.
Previous studies using the enteric bacterial pathogen Salmonella enterica serovar Typhimurium showed that growth in a
ground-based space flight analog bioreactor, termed the rotating
wall vessel (RWV), induced global genotypic and phenotypic
changes in this organism (5–7). Specifically, S. typhimurium
grown in this space flight analog culture environment, described
www.pnas.org"cgi"doi"10.1073"pnas.0707155104
as low-shear modeled microgravity (LSMMG), exhibited increased virulence, increased resistance to environmental stresses
(acid, osmotic, and thermal), increased survival in macrophages,
and global changes in gene expression at the transcriptional and
translational levels (5–7). Collectively, these results suggested
the potential that the true space flight environment could
globally alter bacterial genotypic and phenotypic responses.
Thus, we designed an experimental approach to test our hypothesis, specifically to culture S. typhimurium during space flight and
evaluate changes in microbial gene expression and virulence in
response to this environment.
Our experiments were flown on Space Shuttle Atlantis Mission STS-115 (September 2006). In this experiment, cultures of
S. typhimurium were activated to grow in space for a specific time
period and then either fixed in an RNA/protein fixative or
supplemented with additional growth media after this time
period [supporting information (SI) Fig. 3]. At 2.5 h after landing
at Kennedy Space Center, the culture samples were recovered
and subsequently used for whole-genome transcriptional microarray and proteomic analysis (fixed samples) or for infections
in a murine model of salmonellosis (media-supplemented samples). In each case, the flight culture samples were compared
with culture samples grown under identical conditions on the
ground at Kennedy Space Center using coordinated activation
Author contributions: J.W.W., C.M.O., K.H.z.B., R.R., L.Q., S.P., P.C., M.M., G.T., T.R., A.H.,
D.F., E.R., M.S., M.K., L.J., M.N.-G., S.H., M.P., P.D., K.N., R.B., J.D., A.R., C.G., M.R., L.S., L.C.,
M.J.S., K.B., L.M., J.M., P.A., C.B.-C., T.H., and C.A.N. designed research; J.W.W., C.M.O.,
K.H.z.B., R.R., L.Q., S.P., P.C., M.M., G.T., T.R., A.H., D.F., E.R., M.S., M.K., M.N.-G., S.H., M.P.,
P.D., C.G., M.R., M.J.S., K.B., L.M., J.M., P.A., C.B.-C., T.H., H.M.S.-P., and C.A.N. performed
research; S.P., P.C., M.M., G.T., T.R., A.H., L.J., M.N.-G., L.S., P.S., J.V., R.N., and D.L.P.
contributed new reagents/analytic tools; J.W.W., C.M.O., K.H.z.B., R.R., L.Q., S.P., P.C.,
M.M., G.T., T.R., A.H., D.F., E.R., M.S., M.K., L.J., M.N.-G., P.S., M.J.S., K.B., L.M., P.A., C.B.-C.,
T.H., and C.A.N. analyzed data; and J.W.W., C.M.O., and C.A.N. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: LSMMG, low-shear modeled microgravity; RWV, rotating wall vessel; FPA,
fluid processing apparatus; Km, konamycin.
Data deposition: The data reported in this paper have been deposited in the Gene
Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE8573).
sTo
whom correspondence should be addressed at: The Biodesign Institute, Center for
Infectious Diseases and Vaccinology, Arizona State University, 1001 South McAllister
Avenue, Tempe, AZ 85287. E-mail: cheryl.nickerson@asu.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0707155104/DC1.
© 2007 by The National Academy of Sciences of the USA
PNAS ! October 9, 2007 ! vol. 104 ! no. 41 ! 16299 –16304
MICROBIOLOGY
Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved August 27, 2007 (received for review July 30, 2007)
Fig. 1. Data from STS-115 S. typhimurium experiments. (A) Map of the 4.8-Mb circular S. typhimurium genome with the locations of the genes belonging to
the space flight transcriptional stimulon indicated as black hash marks. (B) Decreased time to death in mice infected with flight S. typhimurium as compared with
identical ground controls. Female BALB/c mice per-orally infected with 107 bacteria from either space flight or ground cultures were monitored every 6 –12 h
over a 30-day period, and the percent survival of the mice in each group is graphed versus the number of days. (C) Increased percent mortality of mice infected
with space flight cultures across a range of infection dosages. Groups of mice were infected with increasing dosages of bacteria from space flight and ground
cultures and monitored for survival over 30 days. The percent mortality (calculated as in ref. 23) of each dosage group is graphed versus the dosage amount. (D)
Decreased LD50 value (calculated as in ref. 23) for space flight bacteria in a murine infection model. (E) SEM of space flight and ground S. typhimurium bacteria
showing the formation of an extracellular matrix and associated cellular aggregation of space flight cells. (Magnification: !3,500.)
and termination times (by means of real-time communications
with the Shuttle crew) in an insulated room that maintained
temperature and humidity identical to those on the Shuttle
(Orbital Environment Simulator). The culture experiments were
loaded into specially designed hardware [termed fluid processing
apparatus (FPA)] to facilitate controlled activation and fixation
of the cultures while maintaining suitable culture containment
requirements (SI Fig. 4).
To our knowledge, our results are the first documentation of
changes in bacterial gene expression and microbial virulence in
response to culture during space flight. Specifically, these findings demonstrate that the space flight environment imparts a
signal that can induce molecular changes in bacterial cells.
Furthermore, these results also provide direct evidence that this
signal can alter the virulence of a microbial pathogen. Our
collective data indicate that the conserved RNA-binding protein
Hfq plays a central regulatory role in the microbial response to
space flight conditions. Evaluation of microbial changes in
response to this unique environment has the potential to provide
heretofore unavailable insight into microbial response mechanisms to Earth-based environments, including those encountered by pathogens during the natural course of infection.
Results
Whole-Genome Transcriptional and Proteomic Analysis of Space Flight
and Ground Cultures. To determine which genes changed expres-
sion in response to space flight, total bacterial RNA was isolated
from the fixed flight and ground samples, qualitatively analyzed
to ensure lack of degradation by means of denaturing gel
electrophoresis, quantitated, and then reverse-transcribed into
labeled, single-stranded cDNA. The labeled cDNA was cohybridized with differentially labeled S. typhimurium genomic
DNA to whole-genome S. typhimurium microarray slides. The
16300 ! www.pnas.org"cgi"doi"10.1073"pnas.0707155104
cDNA signal hybridizing to each gene spot was quantitated, and
the normalized, background-subtracted data were analyzed for
statistically significant 2-fold or greater differences in gene
expression between the flight and ground samples. We found 167
genes differentially expressed in flight as compared with ground
controls from a variety of functional categories (69 up-regulated
and 98 down-regulated) (SI Table 2). The proteomes of fixed
cultures were also obtained by means of multidimensional
protein identification analysis. We identified 251 proteins expressed in the flight and ground cultures, with 73 being present
at different levels in these samples (SI Table 3). Several of the
genes encoding these proteins were also identified by means of
microarray analysis. Collectively, these gene expression changes
form the first documented bacterial space flight stimulon indicating that bacteria respond to this environment with widespread
alterations of expression of genes distributed globally throughout
the chromosome (Fig. 1A).
Involvement of Hfq in Space Flight and LSMMG Responses. Identification of one or more regulators of the space flight stimulon
represents an important step in understanding the nature of this
unique environmental signal. Our data indicated that a pathway
involving the conserved RNA-binding regulatory protein Hfq
played a role in this response (Table 1). Hfq is an RNA
chaperone that binds to small regulatory RNA and mRNA
molecules to facilitate mRNA translational regulation in response to envelope stress (in conjunction with the specialized !
factor RpoE), environmental stress (by means of alteration of
RpoS expression), and changes in metabolite concentrations,
such as iron levels (via the Fur pathway) (8–12). Hfq is also
involved in promoting the virulence of several pathogens including S. typhimurium (13), and Hfq homologues are highly conWilson et al.
Fold
change
Gene
Function
Hfq regulon genes (up-regulated)
Outer membrane proteins
ompA
ompC
ompD
Plasmid transfer apparatus
traB
traN
trbA
traK
traD
trbC
traH
traX
traT
trbB
traG
traF
traR
Various cellular functions
gapA
sipC
adhE
glpQ
fliC
sbmA
2.05
2.44
3.34
Outer membrane porin
Outer membrane porin
Outer membrane porin
4.71
4.24
3.14
2.91
2.87
2.68
2.59
2.37
2.34
2.32
2.21
2.11
1.79
Conjugative transfer
Conjugative transfer
Conjugative transfer
Conjugative transfer
Conjugative transfer
Conjugative transfer
Conjugative transfer
Conjugative transfer
Conjugative transfer
Conjugative transfer
Conjugative transfer
Conjugative transfer
Conjugative transfer
7.67
6.27
4.75
2.58
2.11
1.67
Glyceraldehyde dehydrogenase
Cell invasion protein
Fe-dependent dehydrogenase
Glycerophosphodiesterase
Flagellin, structural protein
ABC superfamily transporter
Hfq regulon genes (down-regulated)
Small RNAs
"RBS
rnaseP
csrB
tke1
oxyS
RFN
rne5
Ribosomal proteins
rpsL
0.305
0.306
0.318
0.427
0.432
0.458
0.499
Small RNA
Small RNA regulatory
Small RNA regulatory
Small RNA
Small RNA regulatory
Small RNA
Small RNA
0.251
30S ribosomal subunit protein
S12
30S ribosomal subunit protein
S19
50S ribosomal subunit protein L4
30S ribosomal subunit protein S6
50S ribosomal subunit protein L16
50S ribosomal subunit protein L1
50S ribosomal protein L31
50S ribosomal subunit protein
L25
rpsS
0.289
rplD
rpsF
rplP
rplA
rpme2
rplY
0.393
0.401
0.422
0.423
0.473
0.551
Various cellular functions
ynaF
ygfE
dps
hfq
osmY
0.201
0.248
0.273
0.298
0.318
mysB
0.341
rpoE
0.403
Wilson et al.
Putative universal stress protein
Putative cytoplasmic protein
Stress response protein
Host factor for phage replication
Hyperosmotically inducible
protein
Suppresses protein export
mutants
!E (!24) factor
Table 1. (continued)
Gene
Fold
change
cspD
nlpb
ygaC
ygaM
gltI
0.421
0.435
0.451
0.453
0.479
ppiB
atpE
yfiA
trxA
nifU
rbfA
rseB
yiaG
ompX
rnpA
hns
lamB
rmf
tpx
priB
0.482
0.482
0.482
0.493
0.496
0.506
0.514
0.528
0.547
0.554
0.554
0.566
0.566
0.566
0.571
Function
Similar to CspA; not cold-induced
Lipoprotein-34
Putative cytoplasmic protein
Putative inner membrane protein
ABC glutamate/aspartate
transporter
Peptidyl-prolyl isomerase B
Membrane-bound ATP synthase
Ribosome-associated factor
Thioredoxin 1, redox factor
Fe-S cluster formation protein
Ribosome-binding factor
Anti-!E factor
Putative transcriptional regulator
Outer membrane protein
RNase P, protein component
DNA-binding protein
Phage # receptor protein
Ribosome modulation factor
Thiol peroxidase
Primosomal replication protein N
Iron utilization/storage genes
adhE
entE
4.76
2.24
hydN
dmsC
nifU
fnr
2.03
0.497
0.495
0.494
fdnH
frdC
bfr
ompW
dps
0.458
0.411
0.404
0.276
0.273
Fe-dependent dehydrogenase
2,3-dihydroxybenzoate-AMP
ligase
Electron transport (FeS center)
Anaerobic DMSO reductase
Fe-S cluster formation protein
Transcriptional regulator,
Fe-binding
Fe-S formate dehydrogenase-N
Fumarate reductase, anaerobic
Bacterioferrin, iron storage
Outer membrane protein W
Stress response protein and
ferritin
Genes implicated in/associated with biofilm formation
wza
wcaI
ompA
wcaD
wcaH
2.30
2.07
2.06
1.82
1.76
manC
wcaG
1.71
1.68
wcaB
fimH
fliS
flgM
flhD
fliE
fliT
cheY
cheZ
1.64
1.61
0.339
0.343
0.356
0.438
0.444
0.461
0.535
Polysaccharide export protein
Putative glycosyl transferase
Outer membrane protein
Putative colanic acid polymerase
GDP-mannose mannosyl
hydrolase
Mannose guanylyltransferase
Bifunctional GDP fucose
synthetase
Putative acyl transferase
Fimbrial subunit
Flagellar biosynthesis
Flagellar biosynthesis
Flagellar biosynthesis
Flagellar biosynthesis
Flagellar biosynthesis
Chemotaxic response
Chemotaxic response
For specific parameters used to identify these genes, please see Materials
and Methods.
PNAS ! October 9, 2007 ! vol. 104 ! no. 41 ! 16301
MICROBIOLOGY
Table 1. Space flight stimulon genes belonging to Hfq regulon
or involved with iron utilization or biofilm formation
served across species of prokaryotes and eukaryotes (14). Our
data strongly support a role for Hfq in the response to space
flight: (i) The expression of hfq was decreased in flight, and this
finding matched previous results in which S. typhimurium hfq
gene expression was decreased in a ground-based model of
microgravity (7). (ii) Expression of 64 genes in the Hfq regulon
was altered in flight (32% of the total genes identified), and the
directions of differential changes of major classes of these genes
matched predictions associated with decreased hfq expression
(see subsequent examples). (iii) Several small regulatory RNAs
that interact with Hfq were differentially regulated in flight as
would be predicted if small RNA/Hfq pathways are involved in
a space flight response. (iv) The levels of OmpA, OmpC, and
OmpD mRNA and protein are classic indicators of the RpoEmediated periplasmic stress response, which involves Hfq (15).
Transcripts encoding OmpA, OmpC, and OmpD (and OmpC
protein level) were up-regulated in flight, correlating with hfq
down-regulation. (v) Hfq promotes expression of a large class of
ribosomal structural protein genes (12), and we found that many
such genes exhibited decreased expression in flight. (vi) Hfq is
a negative regulator of the large tra operon encoding the F
plasmid transfer apparatus (16), and several tra genes from
related operons on two plasmids present in S. typhimurium $3339
were up-regulated in flight. (vii) Hfq is intimately involved in a
periplasmic stress signaling pathway that depends on the activity
levels of three key proteins, RpoE, DksA, and RseB; differential
expression of these genes was observed in flight (8, 12). (viii) Hfq
regulates the expression of the Fur protein and other genes
involved in the iron response pathway, and we observed several
iron utilization/storage genes with altered expression in flight (9,
11). This finding also matched previous results in which iron
pathway genes in S. typhimurium changed expression in a
ground-based model of microgravity, and the Fur protein was
shown to play a role in stress resistance alterations induced in the
same model (7).
Given these findings, we designed experiments to verify a role
for Hfq in the space flight response using a cellular growth
apparatus that serves as a ground-based model of microgravity
conditions termed the RWV bioreactor (SI Fig. 5). Designed by
the National Aeronautics and Space Administration, the RWV
has been extensively used in this capacity to study the effects of
a biomedically relevant low-fluid-shear growth environment
(which closely models the liquid growth environment encountered by cells in the microgravity environment of space flight as
well as by pathogens during infection of the host) on various
types of cells (6, 17–19). Studies with the RWV involve using two
separate apparatuses: one is operated in the low shear modeled
microgravity position (LSMMG), and one is operated as a
control in a position (termed 1 ! g) where sedimentation due to
gravity is not offset by the rotating action of the vessel.
LSMMG-induced alterations in acid stress resistance and macrophage survival of S. typhimurium have previously been shown to
be associated with global changes in gene expression and virulence
(5, 7). We grew WT and isogenic hfq mutant strains of S. typhimurium in the RWV in the LSMMG and 1 ! g positions and
assayed the acid stress response and macrophage survival of these
cultures. Whereas the WT strain displayed a significant difference
in acid resistance between the LSMMG and 1 ! g cultures, this
response was not observed in the hfq mutant, which contains a
deletion of the hfq gene and replacement with a Cm-r cassette (Fig.
2A). Two control strains, hfq 3"Cm (containing an insertion of the
Cm-r cassette just downstream of the WT hfq gene) and invA
kanamycin (Km) (containing a Km-r insertion in a gene unrelated
to stress resistance), gave the same result as the WT strain. We also
observed increased intracellular replication of the LSMMG-grown
WT (hfq 3"Cm) strain in infected J774 macrophages as compared
with the 1 ! g control, and this phenotype was not observed in the
hfq mutant strain (Fig. 2B). Collectively, these data indicate that
16302 ! www.pnas.org"cgi"doi"10.1073"pnas.0707155104
Fig. 2. Hfq is required for S. typhimurium LSMMG-induced phenotypes in
RWV culture. (A) The survival ratio of WT and isogenic hfq, hfq 3"Cm, and invA
mutant strains in acid stress after RWV culture in the LSMMG and 1 ! g
positions is plotted (P # 0.05, ANOVA). (B) Fold intracellular replication of S.
typhimurium strains hfq 3"Cm and $hfq in J774 macrophages after RWV
culture as above. Intracellular bacteria were quantitated at 2 h and 24 h after
infection, and the fold increase in bacterial numbers between those two time
periods was calculated (P # 0.05, ANOVA).
Hfq is involved in the bacterial space flight response as confirmed
in a ground-based model of microgravity conditions. In addition,
the intracellular replication phenotype inside macrophages correlates with the finding that space flight and LSMMG cultures exhibit
increased virulence in mice (see next paragraph and ref. 5).
Increased Virulence of S. typhimurium Grown in Space Flight as
Compared with Ground Controls. Because growth during space
flight and potential global reprogramming of gene expression in
response to this environment could alter the virulence of a
pathogen, we compared the virulence of S. typhimurium space
flight samples to identical ground controls as a second major part
of our study. Bacteria from flight and ground cultures were
harvested and immediately used to inoculate female BALB/c
mice via a per-oral route of infection on the same day as the
Shuttle landing. Two sets of mice were infected at increasing
dosages of either flight or ground cultures, and the health of the
mice was monitored every 6–12 h for 30 days. Mice infected with
bacteria from the flight cultures displayed a decreased time to
death (at the 107 dosage), increased percent mortality at each
infection dosage, and a decreased LD50 value compared with
those infected with ground controls (Fig. 1 B–D). These data
indicate increased virulence for space flight S. typhimurium
samples and are consistent with previous studies in which the
same strain of S. typhimurium grown in the RWV under
Wilson et al.
SEM of Space Flight and Ground Cultures. To determine any mor-
phological differences between flight and ground cultures, SEM
analysis of bacteria from these samples was performed. Although no difference in the size and shape of individual cells in
both cultures was apparent, the flight samples demonstrated
clear differences in cellular aggregation and clumping that was
associated with the formation of an extracellular matrix (Fig.
1E). Consistent with this finding, several genes associated with
surface alterations related to biofilm formation changed expression in flight (up-regulation of wca/wza colonic acid synthesis
operon, ompA, fimH; down-regulation of motility genes) (Table
1). SEM images of other bacterial biofilms show a similar matrix
accumulation (20, 21). Because extracellular matrix/biofilm formation can help to increase survival of bacteria under various
conditions, this phenotype indicates a change in bacterial community potentially related to the increased virulence of the flight
bacteria in the murine model.
Discussion
To our knowledge, these results represent the first documented
gene expression changes that occur in bacterial cells (and any
microbial pathogen) during space flight and accordingly demonstrate that a microgravity growth condition provides an
environmental signal that can induce molecular changes in
bacterial cells. To our knowledge, these results also provide the
first direct evidence that growth during space flight can alter the
virulence of a pathogen; in this study, S. typhimurium grown in
space flight displayed increased virulence in a murine infection
model compared with identical ground controls. Importantly,
these results correlate with previous findings in which the same
strain of S. typhimurium displayed increased virulence in the
murine model after growth in the low-shear microgravity-like
conditions of the RWV bioreactor. In agreement with the
increased virulence observed in the space flight samples, bacteria cultured in flight exhibited cellular aggregation and extracellular matrix formation consistent with biofilm production.
Moreover, several Salmonella genes associated with biofilm
formation changed expression in flight. In addition, the space
flight analogue culture environment of the RWV was used to
verify a mechanistic role for Hfq as a global regulator of
microbial responses during growth in low-shear microgravitylike growth conditions similar to those found in space flight
liquid culture.
Strategies designed to counteract the virulence-enhancing
effects of space flight in microbes provide important potential
benefits to crew health and open insight into novel antimicrobial
strategies on Earth. Accordingly, the identification of global
regulators, such as Hfq, that coordinate microbial responses to
these biomedically relevant environments provides targets at
which these strategies can be directed. Hfq is an RNA-binding
global regulatory protein that is conserved in a wide range of
organisms, both prokaryotes and eukaryotes, and primarily acts
as a chaperone to stabilize interactions between small RNA and
mRNA molecules (14). Further study is needed to determine
whether changes in Hfq regulon expression under space flight
and LSMMG space flight analogue conditions alter critical RNA
interactions that control virulence and other microbial responses. Because low fluid shear is encountered by pathogens in
the host, these responses may be important for bacterial reprogramming during transitions between environments of different
physiological fluid shear levels (such as from the flow of a lumen
to the protected environment between brush border microvilli),
which results in enhanced survival and infection.
Wilson et al.
Materials and Methods
Strains, Media, and Chemical Reagents. The virulent, mousepassaged S. typhimurium derivative of SL1344 termed $3339 was
used as the WT strain in all flight- and ground-based experiments (5). Isogenic derivatives of SL1344 with mutations $hfq,
hfq 3"Cm, and invA Km were used in ground-based experiments
(13, 22). The $hfq strain contains a deletion of the hfq ORF and
replacement with a chloramphenicol resistance cassette, and the
hfq 3"Cm strain contains an insertion of the same cassette
immediately downstream of the WT hfq ORF. The invA Km
strain contains a Km resistance cassette inserted in the invA
ORF. Lennox broth was used as the growth medium in all
experiments (5), and PBS (Invitrogen, Carlsbad, CA) was used
to resuspend bacteria for use as inoculum in the FPAs. The RNA
fixative RNA Later II (Ambion, Austin, TX), glutaraldehyde
(16%; Sigma, St. Louis, MO), and formaldehyde (2%; Ted Pella,
Redding, CA) were used as fixatives in flight experiments.
Loading of FPA. An FPA consists of a glass barrel that can be
divided into compartments (by means of the insertion of rubber
stoppers) and a lexan sheath into which the glass barrel is
inserted (see SI Fig. 4). Each compartment in the glass barrel was
filled with a solution in an order such that the solutions would
be mixed at specific time points in flight via two actions: (i)
downward plunging action on the rubber stoppers and (ii)
passage of the fluid in a given compartment through a bevel on
the side of the glass barrel such that it was released into the
compartment below. Glass barrels and rubber stoppers were
coated with a silicone lubricant (Sigmacote; Sigma) and autoclaved separately before assembly. A stopper with a gasexchange membrane was inserted just below the bevel in the glass
barrel before autoclaving. FPA assembly was performed aseptically in a laminar flow hood in the following order: 2.0 ml of
Lennox broth medium on top of the gas-exchange stopper, one
rubber stopper, 0.5 ml of PBS containing bacterial inoculum
(%6.7 ! 106 bacteria), another rubber stopper, 2.5 ml of either
RNA fixative or Lennox broth medium, and a final rubber
stopper. Syringe needles (gauge 25 5/8) were inserted into rubber
stoppers during this process to release air pressure and facilitate
assembly. To facilitate group activation of FPAs during flight
and to ensure proper containment levels, sets of eight FPAs were
loaded into larger containers termed group activation packs.
Murine Infection Assay. Six- to 8-week-old female BALB/c mice
(housed in the Animal Facility at the Space Life Sciences
Laboratory at Kennedy Space Center) were fasted for %6 h and
then per-orally infected with increasing dosages of S. typhimurium harvested from flight and ground FPA cultures and
resuspended in buffered saline gelatin (5). Ten mice per infectious dosage were used, and food and water were returned to the
animals within 30 min after infection. The infected mice were
monitored every 6–12 h for 30 days. The LD50 value was
calculated by using the formula of Reed and Muench (23).
SEM. A portion of cells from the viable, media-supplemented
cultures from flight and ground FPAs was fixed for SEM analysis
by using 8% glutaraldehyde and 1% formaldehyde and was
processed for SEM as described previously (24).
Microarray Analysis. Total cellular RNA purification, preparation
of fluorescently labeled, single-stranded cDNA probes, probe
hybridization to whole-genome S. typhimurium microarrays, and
image acquisition were performed as previously described (7)
using three biological and three technical replicates for each
culture condition. Flow cytometric analysis revealed that cell
numbers in flight and ground biological replicate cultures were
not statistically different (using SYTO-BC dye per the manuPNAS ! October 9, 2007 ! vol. 104 ! no. 41 ! 16303
MICROBIOLOGY
LSMMG conditions displayed enhanced virulence in a murine
model as compared with 1 ! g controls (5).
facturer’s recommendations; Invitrogen). Data from stored array images were obtained with QuantArray software (Packard
Bioscience, Billerica, MA) and statistically analyzed for significant gene expression differences by using the Webarray suite as
described previously (25). GeneSpring software was also used to
validate the genes identified with the Webarray suite. To obtain
the genes comprising the space flight stimulon as listed in SI
Table 2, the following parameters were used in Webarray: a fold
increase or decrease in expression of 2-fold or greater, a spot
quality (A value) of &9.5, and P value of #0.05. For some genes
listed in Table 1, the following parameters were used: a fold
increase or decrease in expression of value &1.6 or #0.6,
respectively, an A value of 8.5 or greater, and P value of #0.1.
The vast majority of genes listed in Table 1 had an A value of
&9.0 (with most being &9.5) and a P value of 0.05 or less. The
exceptions were as follows: sbmA (P ' 0.06), oxyS (A ' 8.81),
rplY (A ' 8.95), cspD (A ' 8.90), yfiA (P ' 0.08), ompX (P '
0.09), hns (P ' 0.08), rmf (A ' 8.82), wcaD (P ' 0.09), and fliE
(A ' 8.98). To identify space flight stimulon genes also contained in the Hfq regulon, proteins or genes found to be
regulated by Hfq or RNAs found to be bound by Hfq as reported
in the indicated references were scanned against the space flight
microarray data for expression changes within the parameters
above (8, 12, 13, 16, 26).
Multidimensional Protein Identification Analysis via Tandem MS Coupled to Dual Nano-Liquid Chromatography. Acetone–protein pre-
cipitates from whole-cell lysates obtained from flight and ground
cultures (representing three biological replicates) were subjected
to multidimensional protein identification analysis using the
tandem MS–dual nano-liquid chromatography technique as
described previously (27, 28). Tandem MS spectra of peptides
were analyzed with TurboSEQUEST version 3.1 and XTandem
software, and the data were further analyzed and organized by
using the Scaffold program (29, 30). Please see SI Table 3 for the
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Ground-Based RWV Cultures and Acid Stress and Macrophage Survival
Assays. S. typhimurium cultures were grown in the RWV in the
LSMMG and 1 ! g orientations and assayed for resistance to pH
3.5 and survival inside J774 macrophages as described previously
(5), except that the RWV cultures were grown for 24 h at 37°C.
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calculated. A ratio of the percent survival values for the LSMMG
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in the graphs in Fig. 2 were calculated at P # 0.05.
We thank all supporting team members at Kennedy Space Center,
Johnson Space Center, Ames Research Center, Marshall Space Flight
Center, and BioServe Space Technologies; the Crew of STS-115; and
Neal Pellis, Roy Curtiss III, Marc Porter, Brian Haight, Shawn Watts,
Michael Caplan, Joseph Caspermeyer, Clint Coleman, and Charles
Arntzen. This work was supported by National Aeronautics and Space
Administration Grant NCC2-1362 (to C.A.N.), Louisiana Board of
Regents Grant NNG05GH22H (to C.B.-C.), the Arizona Proteomics
Consortium (supported by National Institute on Environmental Health
Sciences Grant ES06694 to the Southwest Environmental Health Sciences Center), National Institutes of Health/National Cancer Institute
Grant CA023074 (to the Arizona Cancer Center), and the BIO5 Institute
of the University of Arizona.
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